Re‐engineering a transferase scaffold for indole C3 methylation in diketopiperazines

The pyrroloindole (hexahydropyrrolo[2,3‐ b ]indole, HPI) structural motif is present in a wide range of natural products with various biological activities, yet its chemical synthesis poses a challenge, particularly regarding methylation at the indole C3 position. In nature, S‐adenosyl methionine (SAM)‐dependent methyltransferases efficiently catalyze this reaction with high stereoselectivity. This study presents the investigation and rational re‐design of a potential methyltransferase, termed SeMT, from the actinomycete Saccharopolyspora erythraea . While its three‐dimensional structure elucidated via X‐ray crystallography confirmed extensive structural similarity to cyclic dipeptide‐processing methyltransferases such as SgMT, its putative catalytic center is clearly divergent. Accordingly, wild‐type SeMT displayed minimal activity with diketopiperazine (DKP) substrates, triggering an extensive mutagenesis effort aimed at iteratively enhancing this methyltransferase function. This work yielded a variant with appreciable activity, which was comprehensively characterized. Notably, a specific mutation within the catalytic triad of SeMT proved critical not only for its own function but also for the temperature‐activity profile of its homolog protein SgMT. Beyond the specific properties of SeMT, these findings hence provide important insights into the active center architecture of indole C3‐methyltransferases, supporting further development of these enzymes into refined biocatalysts for synthetic applications.